C57BL/6小鼠胚胎成纤维细胞生物学特性及饲养层制备

Biological properties of C57BL/6 mouse embryonic fibroblasts and preparation of feeder layers

背景:昆明小鼠胚胎成纤维细胞是目前最常用的饲养层细胞,C57BL/6小鼠胚胎成纤维细胞作为饲养层的研究鲜有报道。目的:体外分离和培养C57BL/6小鼠胚胎成纤维细胞,制备饲养层,力求扩大小鼠胚胎成纤维细胞的来源。方法:用不同浓度胰蛋白酶分步消化法体外分离和培养C57BL/6小鼠胚胎成纤维细胞,观察其生物学特性,研究其生长规律,并制备小鼠胚胎成纤维细胞饲养层,检测干细胞在所制备饲养层上的生长状态。结果与结论:不同浓度胰蛋白酶分步消化法制备的C57BL/6小鼠胚胎成纤维细胞生长状态好,获得的成纤维细胞数量多,增殖活跃。在细胞冻存后1,2周、1,3,6个月内复苏的细胞存活率差异无显著性意义。C57BL/6小鼠胚胎成纤维细胞在第2-5代增殖旺盛,第6代以后细胞增殖出现明显下降。种植到培养皿上的C57BL/6小鼠饲养层细胞在种植后3 d内活力高,种植4 d以后细胞活力急剧下降。所以C57BL/6小鼠胚胎成纤维细胞来源的饲养层的最佳使用时间为灭活后3 d内,C57BL/6小鼠胚胎成纤维细胞饲养层和昆明小鼠胚胎成纤维细胞饲养层一样,能很好地支持胚胎干细胞及诱导多能干细胞生长。

BACKGROUND： Kunming mouse embryonic fibroblasts are the most common feeder layers at present, and there are rare reports addressing C57BL/6 mouse embryonic fibroblasts as feeder layers. OBJECTIVE： To separate and culture C57BL/6 mouse embryonic flbroblasts in vitro, and produce feeder layers to enlarge the resources of mouse embryonic fibroblasts. METHODS： C57BL/6 mouse embryonic fibroblasts were isolated and cultured by trypsin digestion method in vitro The biological characteristics and growth rule of the fibroblasts were investigated, then the feeder layers for the cell culture were produced. The growth of cell colonies on the prepared feeder layer was tested. RESULTS AND CONCLUSION： C57BL/6 mouse embryonic fibroblasts grew well with a large amount, by trypsin digestion method at different concentrations. There was no significance in the survival rate after cryopreservation for I week, 2 weeks, 1 month, 3 months and 6 months. The cells were proliferative from the second to fifth passage and declined sharply after the sixth passage. The planted mouse embryonic fibroblasts feeder layers had a high activity within 3 days, but got a sharp decline after 4 days. So it is best to use C57BL/6 mouse embryonic fibroblast feeder layers within 3 days after they＇re inactivated. C57BL/6 mouse embryonic fibroblast feeder layer can support embryonic stem cells and induce pluripotent stem cells to grow as Kunming mouse embryonic fibroblasts.